DESCRIPTION(provided by applicant): The yeast, Saccharomyces cerevisiae serves as a model eukaryote to study the regulation of phospholipid metabolism/signaling. Diacylglycerol pyrophosphate (DGPP) is a minor phospholipid in yeast that is synthesized from phosphatidate (PA) via the reaction catalyzed by the membrane-associated enzyme PA kinase and is dephosphorylated to PA, and then to diacylglycerol via the reactions catalyzed by the vacuolar membrane-associated DPPI-encoded DGPP phosphatase. The major hypothesis of the work proposed in this application is that DGPP and/or its metabolism plays a role in cellular responses to the stress conditions of zinc deprivation and to oxidative damage in sodM mutants defective in the copper/zinc superoxide dismutase. The DPPI gene is induced by zinc deprivation and is coordinately regulated with other genes in the S. cerevisiae genome that control zinc homeostasis. We propose to examine the hypothesis that the DGPP phosphatase enzyme, whose enzymatic function controls the cellular levels of DGPP and PA, as well as phosphatidylinositol, plays a role in the regulation of phospholipid metabolism/signaling in vacuolar membranes in response to zinc deprivation. We hypothesize that the function of DGPP phosphatase in the vacuolar membrane may be related to functions associated with the copper/zinc superoxide dismutase. Regulation of DGPP phosphatase will be examined in sodldelta mutant cells. The effects of the dppldelta mutation and the overexpression of DGPP phosphatase will be examined in sod1delta mutant cells. Regulation of DGPP phosphatase by phosphorylation via protein kinase A will be examined, and mutant(s) defective in phosphorylation will be used to examine the relevance of DGPP phosphatase phosphorylation in response to stress. The gene encoding PA kinase will be isolated, and a mutant will be constructed and characterized with respect to PA kinase function in response to the stress conditions that regulate DGPP phosphatase. The regulation of PA kinase expression and activity will also be examined. PA kinase will be purified and characterized, and the regulation of activity by phospholipids and water-soluble metabolites will be investigated.